Purification and characterization of three forms of class III alcohol dehydrogenase

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Abstract
We have resolved and characterized three forms of human and rat hepatic class III alcohol dehydrogenase. Separations were carried out in narrow immobilized pH gradients. Both in humans and rats the three forms were visualized by enzyme staining with cinnamol, but not with ethanol. They were insensitive to the inhibitory effect of pyrazole. The isoelectric points were approximately from 6.3–6.4, from 5.9–6.0 and 5.6. Each electroeluted enzyme extract, purified further by analytical isoelectric focusing over the pH range from 5–6 or 6–7, revealed a single band by enzyme and silver staining and by Western blotting followed by avidin‐biotin staining. Polyacryl‐amide gel electrophoresis in the presence of sodium dodecyl sulfate of each extract revealed a single molecular mass species corresponding to class III alcohol dehydrogenase (ADH). All forms of class III alcohol dehydrogenase were recognized by antisera raised against total class III ADH.